Method for treating celiac disease

ABSTRACT

Enteric compositions comprising one or more tight junction agonists and/or one or more tight junction antagonists are provided. Compositions of the invention may comprise a delayed-release coating disposed over a tight junction agonist and/or tight junction antagonist layer which may be disposed over an inert core. Delayed-release coatings may be substantially stable in gastric fluid and substantially unstable in intestinal fluid, thus providing for substantial release of the tight junction agonist and/or antagonist from the composition in the duodenum or jejunum of the small intestine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 12/616,638,filed Nov. 11, 2009, which is a Divisional of application Ser. No.11/673,342, filed Feb. 9, 2007, which claims the benefit of U.S.Provisional Application No. 60/771,454, filed Feb. 9, 2006, each ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention includes pharmaceutical dosage forms comprisingcertain tight junction antagonists or agonists and an enteric coating.

BACKGROUND OF THE INVENTION

Intestinal tight junction dysfunction occurs in a variety of clinicalconditions, including food allergies, infections of the gastrointestinaltract, autoimmune diseases, and inflammatory bowel diseases. Healthy,mature gut mucosa with its intact tight junction serves as the mainbarrier to the passage of macromolecules. During the healthy state,small quantities of immunologically active proteins cross the gut hostbarrier. These proteins are absorbed across the mucosa through at leasttwo pathways. The vast majority of absorbed proteins (up to 90%) crossthe intestinal barrier via the transcellular pathway, followed bylysosomal degradation that converts proteins into smaller,non-immunogenic peptides. Other proteins are transported as intactproteins, through the paracellular pathway, which involves a subtle butsophisticated regulation of intercellular tight junctions that lead toprotein (antigen) tolerance. When the integrity of the tight junctionsystem is compromised, as with prematurity or after exposure toradiation, chemotherapy, and/or toxins, a deleterious response toenvironmental antigens (including autoimmune diseases and foodallergies) can occur.

To meet the many diverse physiological and pathological challenges towhich epithelia are subjected, the tight junctions or zonula occludens(ZO) must be capable of rapid, physiologic, reversible, transient,energy dependent, and coordinated responses that require the presence ofa complex regulatory system.

Zonula occludens toxin, which is produced by Vibrio cholerae, has beencharacterized by Fasano et al., in Proc. Natl. Acad. Sci., USA,8:5242-5246 (1991) and the sequence has been determined (GenBankaccession no. A43864). The ZOT protein of Vibrio cholerae phage CXTΦexploits the physiological mechanisms of tight junction regulation. ZOTpossesses multiple domains that allow a dual function of the protein asa morphogenetic phage peptide for the Vibrio cholerae phage CTXΦ and asan enterotoxin that modulates intestinal tight junctions. When tested onrabbit ileal mucosa, zonulin occludens toxin (ZOT) increased theintestinal permeability by modulating the structure of intercellulartight junctions.

It has been found that ZOT is capable of reversibly opening tightjunctions in the intestinal mucosa, and thus ZOT, when co-administeredwith a therapeutic agent, is able to effect intestinal delivery of thetherapeutic agent, when employed in an oral dosage composition forintestinal drug delivery (WO 96/37196; and U.S. Pat. No. 5,665,389; andFasano et al., J. Clin. Invest., 99:1158-1164 (1997). In U.S. Pat. No.5,864,014, a ZOT receptor has been identified and purified from anintestinal cell line, i.e., CaCo2 cells. Further, in U.S. Pat. No.5,912,323, ZOT receptors from human intestinal, heart and brain tissuehave been identified and purified.

ZOT mediates a cascade of intracellular events by interacting with thesurface of enteric cells. ZOT binding varies within regions of the smallintestine, being detectable in the jejunum and distal ileum, decreasingalong the villous-crypt axis, and not being detectable in the colon.This binding distribution coincides with the regional effect of ZOT onintestinal permeability.

Mammalian proteins that are immunologically and functionally related toZOT have been identified. In U.S. Pat. No. 5,945,510, novel mammalianproteins that are immunologically and functionally related to ZOT andalso function as the physiological modulator of mammalian tightjunctions, have been identified and purified. These mammalian proteins,referred to as “zonulin”, function as the physiological effector ofmammalian tight junctions.

Tight junction agonists (e.g., agonists of ZOT and/or zonulin) ascontemplated herein have been identified that bind to ZOT receptor.These agonists rapidly open tight junctions in a reversible andreproducible manner, and thus can be used to facilitate the intestinalbioavailability of therapeutic or immunogenic agents in the same manneras ZOT is used as an intestinal delivery enhancer, as described in thefollowing patent references: WO 05/010022, WO 96/37196; U.S. Pat. No.5,827,534; U.S. Pat. No. 5,665,389; and U.S. Pat. No. 5,908,825.

Tight junction antagonists (e.g., antagonists of ZOT and/or zonulin) ascontemplated herein have been identified that bind to ZOT receptor, yetdo not function to physiologically modulate the opening of mammaliantight junctions. See, U.S. Pat. No. 6,458,925. The peptide antagonistscompetitively inhibit the binding of ZOT and zonulin to the ZOTreceptor, thereby inhibiting the ability of ZOT and zonulin tophysiologically modulate the opening of mammalian tight junctions.Inhibiting the opening of tight junctions in various anatomical barriersmay be useful in the treatment of autoimmune diseases.

SUMMARY OF THE INVENTION

The present invention provides compositions comprising one or more tightjunction effectors. Such compositions may comprise a core particle, abase coat over the core particle, and a delayed-release coating disposedover the base coat. In some embodiments, the base coat may comprise oneor more tight junction antagonists and/or one or more tight junctionagonists. In some embodiments, the delayed-release coating may besubstantially stable in gastric fluid. In a specific embodiments, thebase coat may comprise one or more tight junction antagonists, one ormore tight junction agonists, or combinations of one or more tightjunction antagonists and tight junction agonists. In some embodiments,the delayed-release coating may comprise a Eudragit L compound.

Typically, the delayed-release coating comprises an enteric agent thatis substantially stable in an acidic environment and substantiallyunstable in a near neutral to alkaline environment. Suitabledelayed-release coatings may comprise one or more triglycerides whichmay be selected from the group consisting of tristearin, triolein,tricaprylin, tricaprin, trimyristin, tripalmitin and trilaurin, and acoating support agent.

An example of a tight junction agonist is zonula occludens toxin (ZOT),which is produced by Vibrio cholerae. A ZOT receptor agonist is acompound which is believed to mediate tight junction opening through thesame receptor utilized by ZOT. In another embodiment, a tight junctionagonist may comprise zonulin. A zonulin receptor agonist is a compoundwhich is believed to mediate tight junction opening through the samereceptor utilized by zonulin. Both ZOT receptor agonists and zonulinreceptor agonists are examples of tight junction agonists. Withoutwishing to be bound by theory, it is believed that ZOT and zonulinutilize the same receptor while functioning as tight junction agonists.In a specific embodiment, compositions of the invention may comprise atight junction antagonist comprising a peptide comprising SEQ ID NO. 1.In another specific embodiment, compositions of the invention maycomprise a tight junction agonist comprising a peptide comprising SEQ IDNO. 2.

Compositions of the invention may comprise one or more therapeuticand/or immunogenic agents. When present a therapeutic and/or animmunogenic agent may be disposed in the core particle, the base coat,and/or the delayed-release coat. Typically, the therapeutic agent and/orimmunogenic agent may be disposed in the base coat and/or the coreparticle. Examples of suitable therapeutic agents include, but are notlimited to, glucose metabolism agents (e.g., insulin, repaglinide,acetohexamide, tolbutamide, chlorpropamide, tolazamide, miglitol,glimepiride, and the like), antibiotics, antineoplastics,antihypertensives, antiepileptics, central nervous system agents, andimmune system suppressants.

In one embodiment, a composition of the invention may comprise aplurality of delayed-release particles present in a tablet, capsule orpowder sachet.

Compositions of the invention may be used to treat a wide variety ofdiseases and medical conditions. In one embodiment, the presentinvention provides a method of treating gastrointestinal inflammationcomprising orally administering to a subject in need thereof apharmaceutical dosage form comprising a composition of the invention. Inanother embodiment, the present invention provides a method for treatinggastrointestinal inflammation in a human who has been examined forgastrointestinal inflammation by a medical practitioner and diagnosed inneed of therapy for the gastrointestinal inflammation treatment, themethod comprising orally administering to the human a pharmaceuticaldosage form comprising a composition of the invention. In anotherembodiment, the present invention provides a method for treatingdiabetes in a human who has been examined for diabetes by a medicalpractitioner and diagnosed in need of therapy for the diabetes, themethod comprising orally administering to the human a pharmaceuticaldosage form comprising a composition of the invention comprising a tightjunction agonist (e.g., a zonulin agonist) and insulin.

In another embodiment, the invention provides a composition comprisingmore that one delayed-release coated particle. Such particles may be thesame or different, for example, may comprise one or more of the same ordifferent tight junction effectors, therapeutic agents and/orimmunogenic agents, and delayed-release coatings. An example of such acomposition might include a first core particle, a first base coat overthe first core particle, wherein the first base coat comprises one ormore tight junction effectors, a first delayed-release coating disposedover the first base coat forming a first delayed-release particle,wherein the first delayed-release coating is substantially stable ingastric fluid; and a second core particle, a second base coat over thesecond core particle, wherein the second base coat comprises one or moretight junction effectors, and a second delayed-release coating disposedover the second base coat forming a second delayed-release particle,wherein the second delayed-release coating is substantially stable ingastric fluid. In some embodiments, the first delayed-release coatingand the second delayed-release coating delay release for differenttimes. For example, about half of the tight junction effector present inthe first delayed-release particle may be released after exposure tointestinal fluid for about 5 minutes to about 10 minutes, and about halfof the tight junction effector present in the second delayed-releaseparticle may be released after exposure to intestinal fluid for about 12minutes to about 18 minutes. In compositions of this type, tightjunction effectors may be distributed in any fashion, for example, thetight junction effector present in the first delayed-release particlemay comprise from about 60% to about 90% by weight of the total tightjunction effector in the composition, and the tight junction effectorpresent in the second delayed-release particle may comprise from about10% to about 40% by weight of the total amount of the tight junctioneffector in the composition. Likewise a first delayed-release particlemay comprises a tight junction antagonist and/or a tight junctionagonist while a second delayed-release particle may comprises a tightjunction antagonist and/or a tight junction agonist. Suitable tightjunction antagonists for compositions of this type include, but are notlimited to, tight junction antagonists comprising SEQ ID NO:1. Suitabletight junction agonists for compositions of this type include, but arenot limited to, tight junction agonists comprising SEQ ID NO:2.Compositions of this type may further comprise one or more therapeuticagents and/or immunogenic agents disposed in any fashion. For example, afirst delayed-release particle and/or a second delayed-release particlemay comprise one or more therapeutic agents and/or one or moreimmunogenic agents. Compositions of this type may comprise a pluralityof first delayed-release particles and/or second delayed-releaseparticles. Compositions of this type may be used to treat a variety ofdiseases and/or medical conditions, for example, the invention providesa method of treating gastrointestinal inflammation comprising orallyadministering to a patient in need thereof the composition comprising afirst delayed-release particle and a second delayed-release particle.

In one specific embodiment, the invention provides a compositioncomprising a core particle, a base coat over the core particle, whereinthe base coat comprises a tight junction antagonist comprising SEQ ID:1and a delayed-release coating disposed over the base coat, wherein thedelayed-release coating is substantially stable in gastric fluid. Asuitable core particle for such a composition may be from about 25 toabout 30 mesh in size. The base coat may comprise a binder (e.g.,Baker's sugar) and the delayed-release coating may comprise EudragitL30D. Any amount of one or more tight junction antagonists may be usedfor example, one or more tight junction antagonists may make up about0.1 wt % to about 20 wt % of the composition, about 1 to about 20 wt %of the composition, about 1 wt % to about 10 wt % of the composition, orabout 4 to about 6 wt % of the composition.

In another specific embodiment, the invention provides a compositioncomprising a core particle, a base coat over the core particle, whereinthe base coat comprises a tight junction agonist comprising SEQ ID:2,and a delayed-release coating disposed over the base coat, wherein thedelayed-release coating is substantially stable in gastric fluid. Asuitable core particle for such a composition may be from about 25 toabout 30 mesh in size. The base coat may comprise a binder (e.g.,Baker's sugar) and the delayed-release coating may comprise EudragitL30D. Any amount of one or more tight junction agonists may be used forexample, one or more tight junction agonists may make up about 0.1 wt %to about 20 wt % of the composition, about 1 to about 20 wt % of thecomposition, about 1 wt % to about 10 wt % of the composition, or about4 to about 6 wt % of the composition. Such compositions may furthercomprise one or more therapeutic agents and/or one or more immunogenicagents. Suitable therapeutic agents include, but are not limited to,glucose metabolism agents (e.g., insulin, repaglinide, acetohexamide,tolbutamide, chlorpropamide, tolazamide, miglitol, glimepiride, and thelike), antibiotics, antineoplastics, antihypertensives, antiepileptics,central nervous system agents, and immune system suppressants. Suitableimmunogenic agents include, but are not limited to, vaccines (e.g.,peptide vaccines, attenuated microorganism vaccines, and/or attenuatedvirus vaccines).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the percentage of tight junction effector releasedas function of time in pH 6 dissolution media dissolution from a twobead formulation prepared in accordance with one embodiment of theinvention.

DESCRIPTION OF THE INVENTION

The invention is directed to compositions comprising one or more tightjunction agonists and/or tight junction antagonists. As used herein,“tight junction effector” may be used to refer collectively to tightjunction agonists and tight junction antagonists. The term “antagonist”is defined as a compound that that prevents, inhibits, reduces orreverses the response triggered by an agonist. Thus, a tight junctionagonist as used herein is a compound that mediates the opening of tightjunctions (e.g., agents that induce physiological, transient disassemblyof tight junctions); a tight junction antagonist is a compound thatprevents, inhibits, reduces or reverses the opening of a tight junctionmediated by a tight junction agonist (e.g., agents that preventphysiological, transient disassembly of tight junctions). In someembodiments, a tight junction agonist may operate by binding to the ZOTand/or zonulin receptor, i.e., may be a ZOT and/or zonulin receptoragonist. In some embodiments, a tight junction antaagonist may operateby binding to the ZOT and/or zonulin receptor, i.e., may be a ZOT and/orzonulin receptor antagonist. Without wishing to be bound by theory, itis believed that ZOT and zonulin modulate tight junction opening throughthe same receptor. In some embodiments, tight junction agonist would notinclude agents that physically disrupt the tight junction structure, forexample, detergents. In some embodiments, a tight junction antagonistwould not include compounds that function by binding to a tight junctionagonist directly. Examples of suitable tight junction effectors include,but are not limited to, peptides comprising one or more of the followingsequences:

-   -   GGVLVQPG (SEQ ID NO:1, a tight junction antagonist sometimes        referred to as AT1001) and FCIGRL (SEQ ID NO: 2, a tight        junction agonist) and functional derivatives of these sequences.

For example, functional derivatives of peptide GGVLVQPG include, but arenot limited to,

(SEQ ID NO: 3) Gly Arg Val Cys Val Gln Pro Gly, (SEQ ID NO: 4)Gly Arg Val Cys Val Gln Asp Gly, (SEQ ID NO: 5)Gly Arg Val Leu Val Gln Pro Gly, (SEQ ID NO: 6)Gly Arg Val Leu Val Gln Asp Gly, (SEQ ID NO: 7)Gly Arg Leu Cys Val Gln Pro Gly, (SEQ ID NO: 8)Gly Arg Leu Cys Val Gln Asp Gly, (SEQ ID NO: 9)Gly Arg Leu Leu Val Gln Pro Gly, (SEQ ID NO: 10)Gly Arg Leu Leu Val Gln Asp Gly, (SEQ ID NO: 11)Gly Arg Gly Cys Val Gln Pro Gly, (SEQ ID NO: 12)Gly Arg Gly Cys Val Gln Asp Gly, (SEQ ID NO: 13)Gly Arg Gly Leu Val Gln Pro Gly, (SEQ ID NO: 14)Gly Arg Gly Leu Val Gln Asp Gly, (SEQ ID NO: 15)Gly Gly Val Cys Val Gln Pro Gly, (SEQ ID NO: 16)Gly Gly Val Cys Val Gln Asp Gly, (SEQ ID NO: 17)Gly Gly Val Leu Val Gln Asp Gly, (SEQ ID NO: 18)Gly Gly Leu Cys Val Gln Pro Gly, (SEQ ID NO: 19)Gly Gly Leu Cys Val Gln Asp Gly, (SEQ ID NO: 20)Gly Gly Leu Leu Val Gln Pro Gly, (SEQ ID NO: 21)Gly Gly Leu Leu Val Gln Asp Gly, (SEQ ID NO: 22)Gly Gly Gly Cys Val Gln Pro Gly, (SEQ ID NO: 23)Gly Gly Gly Cys Val Gln Asp Gly, (SEQ ID NO: 24)Gly Gly Gly Leu Val Gln Pro Gly, (SEQ ID NO: 25)Gly Gly Gly Leu Val Gln Asp Gly, and (SEQ ID NO: 26)Val Asp Gly Phe Gly Arg Ile Gly.

For example, functional derivatives of peptide FCIGRL include, but arenot limited to,

(SEQ ID NO: 27) Xaa₁ Cys Ile Gly Arg Leu, (SEQ ID NO: 28)Phe Xaa₂ Ile Gly Arg Leu, (SEQ ID NO: 29) Phe Cys Xaa₃ Gly Arg Leu,(SEQ ID NO: 30) Phe Cys Ile Xaa₄ Arg Leu, (SEQ ID NO: 31)Phe Cys Ile Gly Xaa₅ Leu, and (SEQ ID NO: 32) Phe Cys Ile Gly Arg Xaa₆.

Xaa₁ may be selected from the group consisting of Ala, Val, Leu, Ile,Pro, Trp, Tyr, and Met; Xaa₂ may be selected from the group consistingof Gly, Ser, Thr, Tyr, Asn, and Gln; Xaa₃ may be selected from the groupconsisting of Ala, Val, Leu, Ile, Pro, Trp, and Met; Xaa₄ may beselected from the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, andGln; Xaa₅ may be selected from the group consisting of Lys and His; andXaa₆ may be selected from the group consisting of Ala, Val, Leu, Ile,Pro, Trp, and Met.

Further functional derivatives of (SEQ ID NO:2) include:

(SEQ ID NO: 33) Xaa₁ Xaa₂ Ile Gly Arg Leu, (SEQ ID NO: 34)Xaa₁ Cys Xaa₃ Gly Arg Leu, (SEQ ID NO: 35) Xaa₁ Cys Ile Xaa₄ Arg Leu,(SEQ ID NO: 36) Xaa₁ Cys Ile Gly Xaa₅ Leu, (SEQ ID NO: 37)Xaa₁ Cys Ile Gly Arg Xaa₆, (SEQ ID NO: 38) Phe Xaa₂ Xaa₃ Gly Arg Leu,(SEQ ID NO: 39) Phe Xaa₂ Ile Xaa₄ Arg Leu, (SEQ ID NO: 40)Phe Xaa₂ Ile Gly Xaa₅ Leu, (SEQ ID NO: 41) Phe Xaa₂ Ile Gly Arg Xaa₆,(SEQ ID NO: 42) Phe Cys Xaa₃ Xaa₄ Arg Leu, (SEQ ID NO: 43)Phe Cys Xaa₃ Gly Xaa₅ Leu, (SEQ ID NO: 44) Phe Cys Xaa₃ Gly Arg Xaa₆,(SEQ ID NO: 45) Phe Cys Ile Xaa₄ Xaa₅ Leu, (SEQ ID NO: 46)Phe Cys Ile Xaa₄ Arg Xaa₆, and (SEQ ID NO: 47) Phe Cys Ile Gly Xaa₅Xaa₆.

Xaa₁ may be selected from the group consisting of Ala, Val, Leu, Ile,Pro, Trp, Tyr, and Met; Xaa₂ is selected from the group consisting ofGly, Ser, Thr, Tyr, Asn, and Gln; Xaa₃ is selected from the groupconsisting of Ala, Val, Leu, Ile, Pro, Trp, and Met; Xaa₄ is selectedfrom the group consisting of Gly, Ser, Thr, Tyr, Asn, Ala, and Gln; Xaa₅is selected from the group consisting of Lys and His; Xaa₆ is selectedfrom the group consisting of Ala, Val, Leu, Ile, Pro, Trp, and Met.

Also contemplated are compositions comprising tight junction effectorstypically classified by one of ordinary skill as small molecules,peptides, peptide mimetics and peptide analogues based on the peptidesof SEQ ID Nos. 1-47. When the tight junction effector is a peptide, anylength of peptide may be used. For example, an effector may be about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14 or about 15 amino acids in length.

In some embodiments, when the effector is a peptide tight junctionagonist, the peptide may be from about 3 to about 12, from about 4 toabout 12, from about 5 to about 12, from about 6 to about 12, from about7 to about 12, from about 8 to about 12, from about 9 to about 12, fromabout 10 to about 12, from about 3 to about 10, from about 4 to about10, from about 5 to about 10, from about 6 to about 10, from about 7 toabout 10, from about 8 to about 10, from about 9 to about 10 amino acidsin length. In some embodiments, when the tight junction effector is atight junction agonist peptide, the peptide may be 9 amino acids or lessin length.

In some embodiments, when the effector is a peptide tight junctionantagonist, the peptide may be from about 3 to about 25, from about 6 toabout 25, from about 8 to about 25, from about 10 to about 25, fromabout 15 to about 25, from about 20 to about 25, from about 6 to about20, from about 8 to about 20, from about 10 to about 20, from about 15to about 20, from about 6 to about 15, from about 8 to about 15, fromabout 10 to about 15, from about 6 to about 10, from about 8 to about10, or from about 9 to about 10 amino acids in length. In someembodiments, a peptide antagonist may be 10 amino acids or less inlength.

The compositions of the invention may be formulated for entericdelivery, for example, may comprise one or more coatings, for example,delayed-release coating containing one or more enteric agents. Adelayed-release coating is typically substantially stable in gastricfluid and substantially unstable (e.g., dissolves rapidly or isphysically unstable) in intestinal fluid, thus providing for substantialrelease of the tight junction effector from the composition in theduodenum or the jejunum. The invention also contemplates that thecomposition may optionally include one or more therapeutic orimmunogenically active molecules.

Compositions of the invention may comprise one or more tight junctioneffectors at a level of from about 0.1 wt % to about 20 wt %, from about0.1 wt % to about 18 wt %, from about 0.1 wt % to about 16 wt %, fromabout 0.1 wt % to about 14 wt %, from about 0.1 wt % to about 12 wt %,from about 0.1 wt % to about 10 wt %, from about 0.1 wt % to about 8 wt%, from about 0.1 wt % to about 6 wt %, from about 0.1 wt % to about 4wt %, from about 0.1 wt % to about 2 wt %, from about 0.1 wt % to about1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.1 wt % toabout 0.8 wt %, from about 0.1 wt % to about 0.7 wt %, from about 0.1 wt% to about 0.6 wt %, from about 0.1 wt % to about 0.5 wt %, from about0.1 wt % to about 0.4 wt %, from about 0.1 wt % to about 0.3 wt %, orfrom about 0.1 wt % to about 0.2 wt % of the total weight of thecomposition. Compositions of the invention may comprise one or moretight junction effectors at a level of about 0.1 wt %, about 0.2 wt %,about 0.3 wt %, about 0.4 wt %, about 0.5 wt %, about 0.6 wt %, about0.7 wt %, about 0.8 wt %, or about 0.9 wt % based on the total weight ofthe composition.

Compositions of the invention may comprise one or more tight junctioneffectors at a level of from about 1 wt % to about 20 wt %, from about 1wt % to about 18 wt %, from about 1 wt % to about 16 wt %, from about 1wt % to about 14 wt %, from about 1 wt % to about 12 wt %, from about 1wt % to about 10 wt %, from about 1 wt % to about 9 wt %, from about 1wt % to about 8 wt %, from about 1 wt % to about 7 wt %, from about 1 wt% to about 6 wt %, from about 1 wt % to about 5 wt %, from about 1 wt %to about 4 wt %, from about 1 wt % to about 3 wt %, or from about 1 wt %to about 2 wt % of the total weight of the composition. Compositions ofthe invention may comprise one or more tight junction effectors at alevel of about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5wt %, about 6 wt %, about 7 wt %, about 8 wt %, or about 9 wt % based onthe total weight of the composition.

The terms “stable in gastric fluid” or “stable in acidic environments”refers to a composition that releases 30% or less by weight of the totaltight junction antagonist or agonist in the composition in gastric fluidwith a pH of 5 or less, or simulated gastric fluid with a pH of 5 orless, in approximately sixty minutes. Compositions of the of theinvention may release from about 0% to about 30%, from about 0% to about25%, from about 0% to about 20%, from about 0% to about 15%, from about0% to about 10%, 5% to about 30%, from about 5% to about 25%, from about5% to about 20%, from about 5% to about 15%, from about 5% to about 10%by weight of the total tight junction antagonist or agonist in thecomposition in gastric fluid with a pH of 5, or less or simulatedgastric fluid with a pH of 5 or less, in approximately sixty minutes. Asuse herein, “about” used to modify a numerical value means within 10% ofthe value. Compositions of the invention may release about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, orabout 10% by weight of the total tight junction antagonist or agonist inthe composition in gastric fluid with a pH of 5 or less, or simulatedgastric fluid with a pH of 5 or less, in approximately sixty minutes.

The term “unstable in intestinal fluid” refers to a composition thatreleases 70% or more by weight of the total tight junction effector inthe composition in intestinal fluid or simulated intestinal fluid inapproximately sixty minutes. The term “unstable in near neutral toalkaline environments” refers to a composition that releases 70% or moreby weight of the total amount of tight junction agonist or antagonist inthe composition in intestinal fluid with a pH of 5 or greater, orsimulated intestinal fluid with a pH of 5 or greater, in approximatelyninety minutes. For example, a composition that is unstable in nearneutral or alkaline environments may release 70% or more by weight of atight junction agonist peptide or a tight junction antagonist peptide ina fluid having a pH greater than about 5 (e.g., a fluid having a pH offrom about 5 to about 14, from about 6 to about 14, from about 7 toabout 14, from about 8 to about 14, from about 9 to about 14, from about10 to about 14, or from about 11 to about 14) in from about 5 minutes toabout 90 minutes, or from about 10 minutes to about 90 minutes, or fromabout 15 minutes to about 90 minutes, or from about 20 minutes to about90 minutes, or from about 25 minutes to about 90 minutes, or from about30 minutes to about 90 minutes, or from about 5 minutes to about 60minutes, or from about 10 minutes to about 60 minutes, or from about 15minutes to about 60 minutes, or from about 20 minutes to about 60minutes, or from about 25 minutes to about 90 minutes, or from about 30minutes to about 60 minutes.

In one embodiment, the delayed-release coating may remain essentiallyintact, or may be essentially insoluble, in gastric fluid. The stabilityof the delayed-release coating can be pH dependent. Delayed-releasecoatings that are pH dependent will be substantially stable in acidicenvironments (pH 5 or less), and substantially unstable in near neutralto alkaline environments (pH greater than 5). For example, thedelayed-release coating may essentially disintegrate or dissolve in nearneutral to alkaline environments such as are found in the smallintestine.

Examples of simulated gastric fluid and simulated intestinal fluidinclude, but are not limited to, those disclosed in the 2005Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or othersimulated gastric fluids and simulated intestinal fluids known to thoseof skill in the art, for example, simulated gastric fluid and/orintestinal fluid prepared without enzymes.

Alternatively, the stability of the delayed-release coating can beenzyme-dependent. Delayed-release coatings that are enzyme dependentwill be substantially stable in fluid that does not contain a particularenzyme and substantially unstable in fluid containing the enzyme. Thedelayed-release coating will essentially disintegrate or dissolve influid containing the appropriate enzyme. Enzyme-dependent control can bebrought about, for example, by using materials which release the activeingredient only on exposure to enzymes in the intestine, such asgalactomannans.

As used herein, tight junction antagonists prevent, inhibit or reducethe opening of tight junctions. Tight junction agonists mediate orfacilitate or augment the opening of tight junctions. Tight junctionantagonists may inhibit the binding of a tight junction agonist (e.g.,ZOT and zonulin) to one or more receptor molecules (e.g., the ZOTreceptor), thereby inhibiting or reducing the ability of the agonist tophysiologically modulate the opening of the tight junctions.

A target organ for the release of the tight junction agonists or tightjunction antagonists from the compositions of the invention is the smallintestine, particularly the duodenum and the jejunum. U.S. Pat. Nos.6,458,925, 5,945,510 and 5,827,534 discuss the possibility of oraldosage compositions for small intestinal delivery of tight junctionantagonists, zonulin or ZOT via gastroresistent tablets or capsules asdescribed in the art. See Remington's Pharmaceutical Sciences, 16th Ed.,Eds. Osol, Mack Publishing Co., Chapter 89 (1980); Digenis et al, J.Pharm. Sci., 83:915-921 (1994); Vantini et al, Clinica Terapeutica,145:445-451 (1993); Yoshitomi et al, Chem. Pharm. Bull., 40:1902-1905(1992); Thoma et al, Pharmazie, 46:331-336 (1991); Morishita et al, DrugDesign and Delivery, 7:309-319 (1991); and Lin et al, PharmaceuticalRes., 8:919-924 (1991) for examples of the preparation of such tabletsor capsules.

In one embodiment, enteric formulations of the invention may utilize oneor more delayed-release coatings and an inert core to provide foreffective, delayed yet substantial delivery of the tight junctionagonist or antagonist compounds together with, optionally, othertherapeutic and/or immunogenic agents. The coated compositions aresubstantially stable in acidic environments or gastric fluid, andsubstantially unstable in near neutral to alkaline or intestinal fluid.Compositions of the invention may also include additional compounds,such as buffers, excipients, talc or binding agents, within the spiritof the invention.

Particles Comprising a Tight Junction Agonist or Antagonist and anEnteric Coating

In one embodiment, the invention provides a composition comprising: acore particle having a base coat comprising one or more tight junctionagonists and/or one or more tight junction antagonists, and adelayed-release coating disposed over the coated core particle. Thedelayed-release coating may be substantially stable in acidicenvironments and/or gastric fluid, and/or substantially unstable in nearneutral to alkaline environments or intestinal fluid thereby exposingthe coated core particle to intestinal fluid. The base coat comprisingone or more tight junction agonists and/or one or more tight junctionantagonists may further comprise one or more therapeutic agents.Optionally a plurality of base coats may be applied to the core each ofwhich may contain a tight junction effector and/or a therapeutic agent.Optionally, the core particle may comprise one or more tight junctioneffectors and/or one or more therapeutic agents.

In one embodiment, a subject in need of treatment may be provided with acomposition as described above in the form of a tablet or capsule thatcontains the coated core particles. Such tablets or capsules may beorally administered. Alternatively, the subject can be provided with apowder sachet comprising the coated core particles and, optionally, oneor more adjuvants such as a sweetener or flavoring agent. The subjectmay then mix the powder with a liquid and orally administer the mixture.

The core particles can include spheres or seeds having an average sizeof from about 5 to about 50 mesh, from about 5 to about 45 mesh, fromabout 5 to about 40 mesh, from about 5 to about 35 mesh, from about 5 toabout 30 mesh, from about 5 to about 25 mesh, from about 5 to about 20mesh, from about 5 to about 15 mesh, from about 5 to about 10 mesh, fromabout 10 to about 50 mesh, from about 10 to about 45 mesh, from about 10to about 40 mesh, from about 10 to about 35 mesh, from about 10 to about30 mesh, from about 10 to about 25 mesh, from about 10 to about 20 mesh,from about 10 to about 15 mesh, from about 15 to about 50 mesh, fromabout 15 to about 45 mesh, from about 15 to about 40 mesh, from about 15to about 35 mesh, from about 15 to about 30 mesh, from about 15 to about25 mesh, from about 15 to about 20 mesh, from about 20 to about 50 mesh,from about 20 to about 45 mesh, from about 20 to about 40 mesh, fromabout 20 to about 35 mesh, from about 20 to about 30 mesh, from about 20to about 25 mesh, from about 25 to about 50 mesh, from about 25 to about45 mesh, from about 25 to about 40 mesh, from about 25 to about 35 mesh,from about 25 to about 30 mesh, from about 30 to about 50 mesh, fromabout 30 to about 45 mesh, from about 30 to about 40 mesh, or from about30 to about 35 mesh. Core particles may be coated with a coatingcomposition, which may include one or more tight junction agonistsand/or one or more tight junction antagonists and/or one or moretherapeutic agents as described herein.

The core particles can be water insoluble particles comprising differentoxides, celluloses, organic polymers and other materials, and mixturesthereof, or water soluble particles comprising different inorganicsalts, sugars, non-pareils and other materials, and mixtures thereof.The core particle may comprise from about 25 to about 75, from about 30to about 75, from about 35 to 75, from about 40 to about 75, from about45 to about 75, from about 50 to about 75, from about 55 to about 75from about 60 to about 75 from about 65 to about 75, from about 70 toabout 75, from about 25 to about 70, from about 25 to about 65, fromabout 25 to about 60, from about 25 to about 55, from about 25 to about50 from about 25 to about 45, from about 25 to about 40, from about 25to about 35, from about 25 to about 30, from about 30 to about 60, fromabout 35 to about 55, from about 40 to about 50, from about 42 to about47, or from about 42 to about 45 wt % of the final particle composition.In one embodiment, the core particle may comprise about 43.2 wt % of thefinal particle composition. Common forms of such core particles arecommercially available such as Celpheres™ or non-pareils. The coreparticles may optionally comprise one or more tight junction effectorcompounds and/or one or more therapeutic agents. Core particles may becoated using techniques known in the art, for example, techniquesdescribed in U.S. Pat. No. 6,248,363 (in particular the Examples) andU.S. Pat. No. 6,294,192 (in particular the examples).

Compositions of the invention can also include one or more of thefollowing formulation aids known to those skilled in the art such as asurface active agent, a filler, a disintegrating agent, an alkalinematerial and/or a binder.

Surface active agents suitable for use in the present invention include,but are not limited to, any pharmaceutically acceptable, non-toxicsurfactant. Classes of surfactants suitable for use in the compositionsof the invention include, but are not limited to polyethoxylated fattyacids, PEG-fatty acid diesters, PEG-fatty acid mono- and di-estermixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oiltransesterification products, polyglycerized fatty acids, propyleneglycol fatty acid esters, mixtures of propylene glycol esters-glycerolesters, mono- and diglycerides, sterol and sterol derivatives,polyethylene glycol sorbitan fatty acid esters, polyethylene glycolalkyl ethers, sugar esters, polyethylene glycol alkyl phenols,polyoxyethylene-olyoxypropylene block copolymers, sorbitan fatty acidesters, lower alcohol fatty acid esters, ionic surfactants, and mixturesthereof. In some embodiments, compositions of the invention may compriseone or more surfactants including, but not limited to, sodium laurylsulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,and triethyl citrate. The surface active agent may be present at a levelof from about 0.1 wt % to about 5 wt % from about 0.1 wt % to about 4.5wt %, from about 0.1 wt % to about 4.0 wt %, from about 0.1 wt % toabout 3.5 wt %, from about 0.1 wt % to about 3.0 wt %, from about 0.1 wt% to about 2.5 wt %, from about 0.1 wt % to about 2.0 wt %, from about0.1 wt % to about 1.5 wt %, from about 0.1 wt % to about 1.0 wt %, fromabout 0.1 wt % to about 0.5 wt %, from about 0.5 wt % to about 5.0 wt %,from about 1.0 wt % to about 5.0 wt % from about 1.5 wt % to about 5.0wt %, from about 2.0 wt % to about 5.0 wt %, from about 2.5 wt % toabout 5.0 wt %, from about 3.0 to about 5.0 wt %, from about 3.5 wt % toabout 5.0 wt %, from about 4.0 wt % to about 5.0 wt %, from about 4.5 wt% to about 5.0 wt %, from about 0.25 wt % to about 2.5 wt %, from about1.0 wt % to about 2.0 wt %, or from about 1.5 wt % to about 2.0 wt %based on the total weight of the final particle composition. In someembodiments, surface active agents may be present in the compositions ofthe invention at a level of about 1.0 wt %, about 1.1 wt %, about 1.2 wt%, about 1.3 wt %, about 1.4 wt %, about 1.5 wt %, about 1.6 wt %, about1.7 wt %, about 1.8 wt %, about 1.9 wt %, or about 2.0 wt % based on thetotal weight of the final particle composition. In one embodiment,compositions of the invention may comprise about 1.8 wt % surfactantbased on the total weight of the final particle composition.

Alkaline material suitable for use in compositions of the inventioninclude, but are not limited to, sodium, potassium, calcium, magnesiumand aluminum salts of acids such as phosphoric acid, carbonic acid,citric acid and other aluminum/magnesium compounds. In addition thealkaline material may be selected from the group consisting of antacidmaterials such as aluminum hydroxides, calcium hydroxides, magnesiumhydroxides and magnesium oxide. The alkaline agent may be present at alevel of from about 1 wt % to about 20 wt %, from about 1 wt % to about18 wt %, from about 1 wt % to about 16 wt %, from about 1 wt % to about14 wt %, from about 1 wt % to about 12 wt %, from about 1 wt % to about10 wt %, from about 1 wt % to about 8 wt %, from about 1 wt % to about 6wt %, from about 1 wt % to about 4 wt %, or from about 1 wt % to about 2wt %, based on the total weight of the coating composition, depending onthe relative strength of the alkaline material. In some embodiments,alkaline material may be present in the compositions of the invention ata level of about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about5 wt %, about 6 wt %, about 7 wt %, about 8 wt %, about 9 wt %, about 10wt %, about 11 wt %, about 12 wt %, about 13 wt %, about 14 wt %, about15 wt %, about 16 wt %, about 17 wt %, about 18 wt %, about 19 wt %, orabout 20 wt % based on the total weight of the coating composition.

Binders suitable for use in the compositions of the invention include,but are not limited to, any pharmaceutically acceptable, non-toxicpharmaceutically acceptable binder. A binder may be a water solublepolymer. In some embodiments a binder may comprise one or more bindersselected from the group consisting of polyvinyl alcohol,polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose,hydroxymethyl cellulose and the like. When a water soluble binder isused, it may be applied from an aqueous medium such as water.Compositions of the invention may comprise one or more binders at alevel of from about 1 wt % to about 15 wt % based on the total weight ofthe coating composition, depending on the relative strength of thealkaline material. Fillers, for example, sugars such as lactose,dextrose, sucrose and maltose, microcrystalline cellulose and the likecan also be included in the coating composition. A preferredcommercially available filler is Bakers Special Sugar.

Before applying the delayed-release coating to the coated core particlethe particle can optionally be covered with one or more separatinglayers comprising pharmaceutical excipients including alkaline compoundssuch as for instance pH-buffering compounds. The separating layeressentially separates the coated core particle from the delayed-releasecoating.

The separating layer can be applied to the coated core particle bycoating or layering procedures typically used with coating equipmentsuch as a coating pan, coating granulator or in a fluidized bedapparatus using water and/or organic solvents for the coating process.As an alternative the separating layer can be applied to the corematerial by using a powder coating technique. The materials forseparating layers are pharmaceutically acceptable compounds such as, forinstance, sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl-cellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures. Additives such asplasticizers, colorants, pigments, fillers, anti-tacking and anti-staticagents, such as for instance magnesium stearate, titanium dioxide, talcand other additives can also be included in the separating layer.

In one embodiment, the delayed-release coating includes an enteric agentthat is substantially stable in acidic environments and substantiallyunstable in near neutral to alkaline environments. A delayed-releasecoating composition can be dispersed or dissolved in either water or ina suitable organic solvent and applied to the core particle by methodswell known to those of ordinary skill in the art. One or moredelayed-release coatings can be applied to the coated core particle.Also, as described, an optional separating layer can be applied to thecoated core particles prior to the application of the delayed-releasecoating.

The enteric agent can be selected from the group consisting of, e.g.,solutions or dispersions of methacrylic acid copolymers, celluloseacetate phthalate, hydroxypropylmethyl cellulose phthalate, polyvinylacetate phthalate, carboxymethylethylcellulose, and Eudragit-typepolymer (poly(methacrylic acid, methylmethacrylate), hydroxypropylmethylcellulose acetate succinate, cellulose acetate trimellitate,shellac or other suitable enteric coating polymers. The Eudragit-typepolymer include Eudragit L, NE, RL, RS. Eudragit L polymers arepreferred. The enteric agent may be a combination of the foregoingsolutions or dispersions.

In another embodiment, the delayed-release coating may degrade as afunction of time when in aqueous solution without regard to the pHand/or presence of enzymes in the solution. Such a coating may comprisea water insoluble polymer. Its solubility in aqueous solution istherefore independent of the pH. The term “pH independent” as usedherein means that the water permeability of the polymer and its abilityto release pharmaceutical ingredients is not a function of pH and/or isonly very slightly dependent on pH. Such coatings may be used to preparesustained release formulations. Suitable water insoluble polymersinclude pharmaceutically acceptable non-toxic polymers that aresubstantially insoluble in aqueous media, e.g. water, independent of thepH of the solution. Suitable polymers include, but are not limited to,cellulose ethers, cellulose esters, or cellulose ether-esters, i.e., acellulose derivative in which some of the hydroxy groups on thecellulose skeleton are substituted with alkyl groups and some aremodified with alkanoyl groups. Examples include ethyl cellulose, acetylcellulose, nitrocellulose, and the like. Other examples of insolublepolymers include, but are not limited to, lacquer, and acrylic and/ormethacrylic ester polymers, polymers or copolymers of acrylate ormethacrylate having a low quaternary ammonium content, or mixturethereof and the like. Other examples of insoluble polymers includeEUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymers usefulin the present invention include polyvinyl esters, polyvinyl acetals,polyacrylic acid esters, butadiene styrene copolymers, and the like.

In another embodiment, the delayed-release coating contains an entericagent that is substantially stable in gastric fluid. The enteric agentwill be sensitive to pancreatic lipase. The enteric agents sensitive topancreatic lipase include the triglycerides, tristearin, triolein,tricaprylin, tricaprin, trimyristin, tripalmitin and trilaurin. One ormore of these triglycerides in combination with a coating support agent,e.g., a cellulose-type material such as ethylcellulose, are used toprepare a delayed relay coating composition, which is then applied tothe coated core particles.

A delayed-release coating composition may comprise from 0.1 wt % to 5 wt% of a triglyceride or a mixture of triglycerides, and 0.5 wt % to 10 wt% of a coating support agent. Another delayed-release coatingcomposition will comprise from 0.5 wt % to 3 wt % of a triglyceride or amixture of triglycerides, and 1 wt % to 5 wt % of a coating supportagent.

One or more delayed-release coatings can be applied to the coated coreparticle. For example, an additional delayed-release coatings can beapplied to the coated core particle in addition to an enteric agentsensitive to pancreatic lipase. Such an additional coating can includean enteric agent that is substantially stable in an acidic environmentas described herein. Also, as described, an optional separating layercan be applied to the coated core particles prior to the application ofthe delayed-release coating.

The delayed-release coating compositions can also include one or moreinert processing aids in an amount from 10 to 80 wt % based on the totalweight of the coating composition. The inert processing aids includefinely divided forms of talc, silicon dioxide, magnesium stearate andthe like.

The delayed-release coating compositions can also containpharmaceutically acceptable plasticizers to obtain the desiredmechanical properties such as flexibility and hardness. Suchplasticizers include, but are not limited to, triacetin, citric acidesters, phthalic acid esters, dibutyl sebacate, cetyl alcohol,polyethylene glycols, polysorbates or other plasticizers.

The amount of plasticizer is optimized for each delayed-release coatingin relation to the selected enteric agent used. A selected plasticizerand the applied amount of enteric agent are optimized for the desiredmechanical properties, i.e., flexibility and hardness of thedelayed-release coating. The hardness of the delayed-release coating isoften exemplified as Vickers hardness, and is adjusted so that the acidresistance of the final coated particle does not decrease significantlyduring the compression of the particles into tablets. Other compoundscan be added to the delayed-release coating composition to increase filmthickness and to increase the resistance to acidic gastric juices in thestomach.

The delayed-release coating compositions can also include one or moreapplication solvents. Some of the more common solvents that can be usedto apply the delayed-release coating composition include isopropylalcohol, acetone, methylene chloride and the like. Generally the entericagent and inert processing aids will account for 5 wt % to 60 wt % ofcoating composition including the weight of the solvent.

The coated particle with the delayed-release coating can further becovered with an overcoat layer. The overcoat layer can be applied asdescribed for the other coating compositions. The overcoat materials arepharmaceutically acceptable compounds such as sugar, polyethyleneglycol, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate,hydroxypropyl cellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose sodium and others, used alone orin mixtures. The overcoat materials can prevent potential agglomerationof particles coated with the delayed-release coating, protect thedelayed-release coating from cracking during the compaction process orenhance the tableting process.

It is understood that the overall release profile of the final productmay be adjusted by combining two or more particle types with differentrelease profiles. For example, in one embodiment, about half of thecoated core particles are a first coated core particle type that has anenteric coating that exposes the tight junction agonist or antagonist tointestinal fluid after about 5 minutes to about 10 minutes, and abouthalf of the coated core particles are a second coated core particle typethat has an enteric coating that exposes the tight junction agonist orantagonist to intestinal fluid after about 12 minutes to about 18 to 60minutes. In another embodiment, the first coated particles comprise from60% to 90% by weight, and the second coated particles comprise 10% to40% by weight, of the total amount of the tight junction effector in thecomposition.

Compositions Comprising One or More Tight Junction Antagonists

In on embodiment, compositions of the invention may comprise one or moretight junction antagonists. An example of suitable antagonists ofzonulin are peptide GGVLVQPG (SEQ ID NO: 1) and derivatives thereof,particularly derivatives having one or more conservative amino acidsubstitutions. Such compositions may be used to treat a wide variety ofdiseases including, but not limited to, autoimmune diseases. Examples ofautoimmune diseases that can be treated using the compositions of theinvention include, but are not limited to, celiac disease, primarybiliary cirrhosis, IgA nephropathy, Wegener's granulomatosis, multiplesclerosis, scleroderma, systemic sclerosis, type 1 diabetes mellitus,rheumatoid arthritis, Crohn's disease, lupus erythematosus, Hashimoto'sthyroiditis (underactive thyroid), Graves' disease (overactive thyroid),autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid,Devic's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenicsyndrome (LEMS), autoimmune lymphproliferative syndrome (ALPS),paraneoplastic syndromes, polyglandular autoimmune syndromes (PGA), andalopecia greata.

Compositions of the invention can be used as anti-inflammatory agentsfor the treatment of gastrointestinal inflammation that gives rise toincreased intestinal permeability. Thus, the antagonists are useful,e.g., in the treatment of intestinal conditions that cause proteinlosing enteropathy. Protein losing enteropathy may arise due to:

-   -   infection, e.g., C. difficile infection, enterocolitis,        shigellosis, viral gastroenteritis, parasite infestation,        bacterial overgrowth, Whipple's disease;    -   diseases with mucosal erosion or ulcerations, e.g., gastritis,        gastric cancer, collagenous colitis, inflammatory bowel disease;        and    -   mucosal diseases without ulceration, e.g., Menetrier's disease,        celiac disease, eosinophilic gastroenteritis.

Other diseases that may be treated with the compositions and methods ofthe invention include, but are not limited to, diseases marked bylymphatic obstruction, e.g., congenital intestinal lymphangiectasia,sarcoidosis lymphoma, mesenteric tuberculosis, and after surgicalcorrection of congenital heart disease; and immune diseases, e.g.,systemic lupus erythematosus or food allergies, primarily to milk (seealso Table 40-2 of Pediatric Gastrointestinal Disease PathophysiologyDiagnosis Management, Eds. Wyllie et al, Saunders Co. (1993), pages536-543; which is incorporated by reference herein in its entirety).

The pharmaceutically effective amount of an antagonist of zonulin willvary depending upon the disease or condition being treated, as well asthe age, weight and sex of the subject being treated. Generally, theamount of antagonist used to inhibit gastrointestinal inflammation,e.g., to inhibit zonulin biological activity, is in the range of about1.0 μg to 1000 μg, preferably about 1.0 μg to 100 μg.

The peptide tight junction antagonists can be chemically synthesized andpurified using well-known techniques, such as described in HighPerformance Liquid Chromatography of Peptides and Proteins: SeparationAnalysis and Conformation, Eds. Mant et al, C.R.C. Press (1991), and apeptide synthesizer, such as Symphony (Protein Technologies, Inc); or byusing recombinant DNA techniques, i.e., where the nucleotide sequenceencoding the peptide is inserted in an appropriate expression vector,e.g., an E. coli or yeast expression vector, expressed in the respectivehost cell, and then purified using well-known techniques.

Compositions Comprising One or More Tight Junction Agonists

In one embodiment, a tight junction agonist may comprise a peptidesequence. Examples of suitable peptide sequences are FCIGRL (SEQ ID NO:2) and derivatives thereof, particularly derivatives having one or moreconservative amino acid substitutions. A peptide sequence may be usedalone or may be part of a larger molecule, e.g., a polypeptide. When anagonist of zonulin comprises a polypeptide, such a polypeptide istypically less than 100 amino acid residues in length, or less than 50,40, 30, 20, 10, or 8 amino acid residues. The polypeptide may containonly the six amino acids FCIGRL (SEQ ID NO: 2), or it can includeadditional amino acids. The other amino acids may provide otherfunctions, e.g., antigen tags, for facilitating purification.

In one embodiment, compositions of the invention may comprise one ormore tight junction agonists and one or more therapeutic and/orimmunogenic agents. In one embodiment, a composition of the inventionmay comprise a therapeutic and/or an immunogenic agent for treating,ameliorating or preventing one or more diseases and an agonist of ahuman receptor of zonulin and vibrio cholerae phage CTXΦ ZOT protein. Itis also contemplated that the composition can exist in the form of apharmaceutical kit in which the agonist and therapeutic agent areassociatively packaged for simultaneous, nearly simultaneous orsubsequent administration. In the later instance, the agonist andtherapeutic agent are administered within 12, 8, 4, 2, or 1 hours ofeach other or as little as within 5, 10 or 15 minutes of each other.

Therapeutic agents that can be used in the compositions include agentsthat act on any organ of the body, such as heart, brain, intestine, orkidneys.

The particular therapeutic or immunogenic agent used in the compositioncan be, any small molecule compound, biologically active peptide,vaccine, or any other moiety otherwise not adequately absorbed throughthe transcellular pathway, regardless of size or charge.

Examples of drug compounds which can be employed in the presentinvention include, but are not limited to, drugs which act on thecardiovascular system, drugs which act on the central nervous system,antineoplastic drugs and antibiotics. Examples of drugs which act on thecardiovascular system include, but are not limited to,antihypertensives, statins, adenosine, dobutamine, dopamine,epinephrine, norepinephrine, and phentolamine. Others as are known inthe art can also be used.

Examples of drugs which act on the central nervous system include, butare not limited to, doxapram, alfentanil, dezocin, nalbuphine,buprenorphine, naloxone, ketorolac, midazolam, and propofol. Otherexamples include, but are not limited to, antipsychotics,antidepressants, antiepileptics, and drugs used to treat Alzheimersdisease. Others as are known in the art can also be used.

Examples of antineoplastic drugs include, but are not limited to,cytarabine, mitomycin, doxorubicin, vincristine and vinblastine,carboplatin, cisplatin, oxaloplatin, vinorelbine, docetaxel, paclitaxel,taxane, 5-fluorouridine related drugs, xeloda, germcitabine, andanthracline. Additional examples include, but are not limited to,Erbitux, Herceptin®, Avastin™, and estrogen receptor antagonists andagonists. Others as are known in the art can also be used.

Examples of antibiotics include, but are not limited to, methicillin,mezlocillin, piperacillin, cetoxitin, cefonicid, cefinetazole andaztreonam. Others as are known in the art can also be used.

Any type of therapeutic and/or immunogenic agent can be used in thepractice of the invention. Examples of specific types of agents include,but are not limited to, RNAi, treatment aptamers, antivirals (e.g.,amantadine, rimantadine, zanamavir and oseltamivir), immune suppressants(e.g., cyclosporine A), HIV fusion inhibitors (e.g., enfuvirtide), andHIV protease inhibitors, (e.g., ritonavir, saquinavir, indinavir,amprenavir, nelfinavir, lopinavir, atazanavir, entricitabine, andfosamprenavir calcium).

Examples of biologically active peptides include hormones, lymphokines,globulins, and albumins. Examples of hormones which can be employed inthe present invention include testosterone, nandrolene, menotropins,insulin, growth hormone, parathyroid hormone (PTH) and urofolltropin.Others as are known in the art can also be used. If the biologicallyactive ingredient is insulin, the oral dosage composition is useful forthe treatment of diabetes. Examples of lymphokines which can be employedin the present invention include interferon-α, interferon-β,interferon-γ, interleukin-1, interleukin-2, interleukin-4 andinterleukin-8.

Examples of globulins include α-globulins, β-globulins and γ-globulins(immunoglobulin). Examples of immunoglobulins which can be employed inthe present invention include polyvalent IgG or specific IgG, IgA andIgM, e.g., anti-tetanus antibodies. An example of albumin which can beused is human serum albumin. Others as are known in the art can also beused.

Examples of vaccines that can be used in the compositions includepeptides and attenuated microorganisms and viruses. Examples of peptideantigens include the B subunit of the heat labile enterotoxin ofenterotoxigenic E. coli, the B subunit of cholera toxin, capsularantigens of enteric pathogens, fimbriae or pili of enteric pathogens,HIV surface antigens, dust allergens, and acari allergens. Otherimmunogenic compounds as are known in the art can also be used.

Examples of attenuated microorganisms and viruses that can be used inthe compositions include those of enterotoxigenic Escherichia coli,enteropathogenic Escherichia coif, hibrio cholerae, Shigella flexneri,Salmonella typhi and rotavirus (Fasano et al, In: Le Vaccinazioni inPediatria, Eds. Vierucci et al, CSH, Milan, pages 109-121 (1991);Guandalini et al, In: Management of Digestive and Liver Disorders inInfants and Children, Elsevior, Eds. Butz et al, Amsterdam, Chapter 25(1993); Levine et al, Sem. Ped. Infect. Dis., 5.243-250 (1994); andKaper et al, Clin. Micrbiol. Rev., 8:48-86 (1995), each of which isincorporated by reference herein in its entirety).

The pharmaceutical compositions of the invention can be used fortreating, ameliorating, and/or preventing a disease. The disease can beselected from the group consisting of cancer, autoimmune, vascular,bacterial infection, gastritis, gastric cancer, collagnenous colitis,inflammatory bowel disease, osteoporosis, systemic lupus erthtematosus,food allergy, asthma, and irritable bowel syndrome.

In another embodiment, the composition can be used in a method oftreating a patient with increased expression of zonulin relative to acontrol healthy individual. In this case, the therapeutic agent may bean antibody that was raised against amino acids SLIGKVDGTSHVTG (SEQ IDNO: 48). For example, the antibody can bind to a protein expressed inCaCo2 cells that co-localizes with a protein bound by syntheticinhibitor peptide SEQ ID NO: 1. The antibody does not bind to human orrat cells that express a recombinant human PAR-2. The antibody is notSAMl1.

It is to be understood, that for any of the antagonist and agonistpeptides described in this application one or more conservativesubstitutions can be made in which an amino acid is exchanged foranother having similar properties. Conservative substitutions of certainamino acids can be made in the agonist peptides of zonulin as well asthe antagonist peptides of zonulin. For example, a conservativesubstitution can be made in the agonist peptide having the sequence ofany one of SEQ ID NO: 1-47. Examples of conservative substitutionsinclude, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr. Conservative amino acid substitutions typically fall in the rangeof about 1 to 2 amino acid residues.

Guidance in determining which amino acid residues can be substitutedwithout abolishing biological or immunological activity can be foundusing computer programs well known in the art, such as DNASTAR software,or in Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure(Natl. Biomed. Res. Found., Washington, D.C.).

Amino acid substitutions are defined as one for one amino acidreplacements. They are conservative in nature when the substituted aminoacid has similar structural and/or chemical properties. Examples ofconservative replacements are substitution of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine.

Particularly preferred oligopeptide analogs include substitutions thatare conservative i.e., those substitutions that take place within afamily of amino acids that are related in their side chains.Specifically, amino acids are generally divided into families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptopban; (4) uncharged polar—glycine,asparagine, glutamae, cysteine, serine threonine, and tyrosine; and (5)aromatic amino acids—phenylalanine, tryptophan, and tyrosine. Forexample, it is reasonably predictable that an isolated replacement ofleucine with isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar conservative replacement of anamino acid with a structurally related amino acid, will not have a majoreffect on the biological activity.

The compositions described can be administered one to several times aday. The typical daily dose of the agonist or antagonist of zonulinvaries and will depend on various factors such as the individualrequirements of the patients, the mode of administration and disease. Inone embodiment, antagonists of zonulin are administered prior to eating.Applications of such administrations include the treatment of Celiacdisease.

In general the daily dose of the agonist or antagonist of ZOT and/orzonulin will be in the range of 1-1000 mg of active substance.

EXAMPLES Example 1 Preparation of a Delayed-Release Particle Comprisinga Tight Junction Antagonist

A base coat containing the tight junction antagonist of SEQ ID NO: 1 wasprepared by mixing 2000 g of water, slowly adding 15 g of the antagonistpeptide. Once the peptide was dispersed, 15 g of Bakers Special Sugarwas added. Total weight was adjusted to 2107 g with additional water.One skilled in the art will appreciate that binders other than BakersSpecial Sugar may be used. Suitable binders are typicallypharmaceutically acceptable binders including, but not limited to,starch, gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes andthe like. Other suitable binders include pharmaceutically acceptablebinders such as polyvinyl pyrrolidone, hydroxypropyl cellulose,hydroxyethyl cellulose, ethylcellulose, polymethacrylate, waxes and thelike. Mixtures of the aforementioned binders may also be used.

After complete dissolution, the base coat is applied. The quantity ofbase coat used is determined by the final weight of enteric-coated beadsdesired. For the amount described above, 3270 g of the base coat wasadded to a Wurster unit charged with 1000 g Non-pareils 25/30 mesh (aratio of 3.27 g base coat/gram non-pareils) under the followingconditions until coating was complete: 135-150° F., Process Air Volume(SCFM) of 10-25 adjusted to maintain fluidization; spray rate of 3-10g/min/nozzle; and atom air pressure of 25 psi.

A delayed-release coating containing Eudragit L30D was also prepared. 15g of Talc was added to 500 g of water and stirred vigorously for 10minutes. 850 grams of Eudragit L30D (Type C) was sifted through a 60mesh screen into the mixing container by vortexing without entrainmentof air. 30 g of triethyl citrate Pharmaceutical Grade was then addedslowly and mixed for 10 minutes under similar conditions and mixingcontinued until completed dissolution and dispersion occurred. Totalmass was brought to 1500 g with the addition of water.

The delayed-release coating was then added to Wurster unit (containingthe base coated particles) under the following conditions until coatingwas complete: 135-150° F., Process Air Volume (SCFM) of 10-25 adjustedto maintain fluidization; spray rate of 3-10 g/min/nozzle); and atom airpressure of 25 psi.

The final composition was as follows:

TABLE 1 Component wt % Non-pareils 25/30 43.2 Antagonist 1 BakersSpecial Sugar 1 Talc 0.9 Eudragit L30D 53.1 Triethyl Citrate 1.8

Example 2

The stability of delayed-release particles containing 4-6% tightjunction antagonist peptide (SEQ ID NO: 1) was performed.Delayed-release particles prepared as described above were exposed toSimulated Gastric Fluid (SGF) containing 0.1% v/v Tween 80K undercontrolled temperature (37° C.±0.5° C.) and agitation in a dissolutionapparatus for 1 hour. The beads were removed from the SGF and exposed toSimulated Intestinal Fluid (SIF) containing 0.1% v/v Tween 80K. Sampleswere pulled from the bath and analyzed by HPLC at 220 nm on a InertsilODS-2 HPLC Column (150 mm×3 mm, 5 μm) with an Inertsil ODS-2 HPLC GuardColumn (20 mm×4.6 μm, 5 μm). Mobile Phase A: 92:8 Water:ACN, 0.1% TFAMobile Phase B: ACN, 0.1% TFA). The chromatographic conditions are shownbelow.

Time Flow rate % H₂O % CH3CN (min) (mL/min) 0.1% TFA 0.1% TFA 0 1.5 98 210 1.5 53.4 46.6 10.1 1.5 0 100 11.9 1.5 0 100 12 1.5 98 2 17 98 2 Runtime: 17 min Detection: 220 nm Response time: 0.5 sec Column Temp.: 40°C. ± 1° C. Auto Sampler Temp: 10° C. ± 2° c. Injection Volume: 5 μl ofsolution

The percentage of total tight junction effector released is determinedby comparison to a reference standard of the effector.

Over a time course of 60 minutes, less than 8% of the antagonist peptideof SEQ ID NO:1 was released in the presence of simulated gastric fluid(SGF) containing 0.1% v/v Tween 80K.

Simulated Gastric Fluid may be prepared as follows: per liter, place thefollowing into a container: 2.0 gram sodium chloride, 7.0 mLconcentrated hydrochloric acid, and 1.0 L water. Stir until completelydissolved. Check the pH and adjust to 1.2±0.1 with dilute HCl or NaOH.De-aerate medium. To this solution Tween 80 was added to 0.1% (v/v).Alternatively, commercially available SGF may be used (Ricca ChemicalCompany, Arlington, Tex.) and Tween 80K added.

Over 90% of the antagonist peptide of SEQ ID:1 was released in thepresence of simulated intestinal fluid (SIF) containing 0.1% v/v Tween80K within 15 minutes and essentially all of the peptide was releasedwithin 60 minutes. SIF can be prepared as follows: place the followinginto a container: 1.9 Kg distilled water, 136 g monobasic potassiumphosphate, and 18.1 gram of sodium hydroxide. Stir until completelydissolved. Dilute the solution with 18.0 Kg water. Check the pH andadjust to 6.8±0.1 with dilute HCl or NaOH. De-aerated medium. To thissolution add Tween 80K to 0.1% (v/v). Alternatively, commerciallyavailable SIF may be purchase (Ricca Chemical Company, Arlington Tex.)and Tween 80K added to 0.1% (v/v).

Example 3 Preparation of Multiparticulate Enterically Coated BeadFormulation for Delayed-Release of Tight Junction Effectors

This embodiment provides for encapsulaton of a combination of aplurality (e.g., 2, 3, 4, 5 etc) of different enterically coated beads.Any amount of each type of bead may be encapsulated. In someembodiments, two different beads may be encapsulated in any proportion.In one specific embodiment, almost equal proportions of two beads may beencapsulated. Each bead may be coated with any suitable coating materialat any suitable coating level. In some embodiments, two beads may becoated with Eudragit L30 D55 at 20 and 70% coating levels. A formulationcomprising 20% and 70% coating levels allows for the delayed-release ofone or more tight junction effectors (e.g. AT1001) into both theduodenum and jejunum. For calculation of weight percentage, weightmeasurements are done before the two substances are put together. Forexample, a 70% coating level is 70 g coating polymer added to 100 gbead.

Eudragit L30D is a polymeric coating that starts to degrade at pH>5.5.Therefore beads coated with Eudragit L30D55 are designed to remainintact as they pass through the stomach but start to dissolve as soon asthey enter the duodenum. This is the case for the beads coated with 20%Eudragit L30D55. Beads coated with 70% Eudragit L30D55 dissolve about 30minutes later in pH 6.0 dissolution media and are designed to startreleasing as soon as they enter the jejumun. FIG. 1 shows a dissolutionprofile of beads prepared according to this embodiment.

Essentially beads may be prepared in two steps. First drug is layeredonto non-pareil beads. Then the drug layered beads may be entericallycoated with L30D55 to the desired level.

It was found that coating less than 20% Eudragit L30D55 started todissolve in vitro in simulated gastric fluid (pH 1.1) and therefore willnot remain undissolved in the stomach. Coating more than 70% was notpossible due to limitations of the coating material.

In one embodiment, beads may be polymethacrylate which are a 1:1 ofpolymethacrylic acid and ethyl acetate (e.g., Eudragit L30D55).

Example 4

In some embodiments, various parameters may be modified in order tooptimize AT1001 enteric coated bead formulation with the goal ofimproving stability of the tight junction effector (both chemical anddissolution stability), for example, as measured at 40° C./75% relativehumidity conditions.

In some embodiments, one or more additional coatings may be applied tothe formulations of the invention. For example, one or more seal coatsand/or top coats may be used. In a specific embodiment, a seal coat maybe applied to the drug layer. In embodiments of this type, a coreparticle may be coated with tight-junction-effector-containing layer,which may be further coated with a sealing coat. After application ofthe sealing coat, one or more delayed-release coating (e.g., EudragitL30D55) may be applied. After the delayed-release coating is applied, asealing coat may be applied. Any suitable sealing coat known to thoseskilled in the art may be used. Examples of suitable sealing coats,include, but are not limited to, HPMC (hydroxypropylmethylcellulose)such as Seppifilm™, Opadry®AMB (polyvinyl alcohol and lecithin), andKollicoat® Protect (polyvinyl alcohol-polyethylene glycol copolymer andpolyvinyl alcohol). Any other suitable sealing coat known to thoseskilled in the art may be used.

In some embodiments, formulations of the invention may comprise a topcoat around the outside of the bead. These formulations may alsocomprise a sealing coat. For example, an embodiment of this type mightcomprise a core particle, a drug layer, a sealing coat, a polymer coatand a top coat.

In some embodiments of the invention, the drug layer may comprise anon-reducing sugar (e.g., trehalose) as a binder. A formulation of thistype might comprise a core particle, atight-junction-effector-containing layer that also comprises trehalose,and a delayed-release polymer layer. Such a formulation might furthercomprise a sealing layer over the tight-junction-effector-containinglayer and/or a top coat layer over the delayed-release layer asdescribed above.

The delayed-release coat (e.g., a coat comprising Eudragit L30D55) maybe formulated using any techniques known in the art. In someembodiments, the delayed-release coating may comprise talc. Talc may bepresent as from about 0.5% to about 15%, from about 1% to about 15%,from about 2% to about 15%, from about 3% to about 15%, from about 4% toabout 15%, from about 5% to about 15%, from about 6% to about 15%, fromabout 7% to about 15%, from about 8% to about 15%, from about 9% toabout 15%, from about 10% to about 15%, from about 11% to about 15%,from about 12% to about 15%, from about 13% to about 15%, from about 14%to about 15% by weight of the solution used to apply the coat. Thus, thesolution used to apply the delayed-release coat may comprise about 0.5%,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14% or about 15% talc by weight.

Formulations of the invention may further comprise one or more additivesthat may be applied to the beads after the application of adelayed-release coat, after the application of any top coat, after thedrug layer, or in lieu of the enteric coat, all as non-limitingexamples. Such additives may comprise one or more agents to impartdesired dissolution or other characteristics, non-limiting examples ofwhich as disclosed in Asghar et al, J Pharm Pharm Sci (2006) 9(3) pages327-338. Such examples include means of obtaining different pH-dependentrelease (such as Eudragit L-100, Eudragit S-100, cellulose acetatephthalate (CAP), shellac, ethyl cellulose), time-dependent release (e.g.hydroxy propyl methyl cellulose, hydroxyethyl cellulose,microcrystalline cellulose, lactose/behinic acid, hydroxy propyl methylcellulose, hydroxy propyl methyl cellulose acetate succinate) or releasedependent on bacteria (e.g. chitosan, pectin, guar gum, chondroitinsulphate, amylose, alginates). Other additives may include agents tomake the mixture more uniform, for example, silicon dioxide or syloid.All of the above are non-limiting examples of agents known in the art

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for treating a celiac patient,comprising: administering a peptide having the amino acid sequence ofSEQ ID NO:1 to the duodenum of the patient; wherein the peptide isformulated as an oral dosage, delayed-release composition that containspeptide-coated beads having a delayed release coating that is a 1:1co-polymer of methacrylic acid and ethyl acrylate and which is stable ingastric fluid and unstable in near neutral to alkaline environments soas to release peptide in the duodenum, and wherein at least 70% of thepeptide in said beads is released upon exposure for 90 minutes tosimulated intestinal fluid having a pH of greater than 5, and whereinthe beads contain 0.1 to 0.8% by weight of said peptide.
 2. The methodof claim 1, wherein the composition is an oral dosage composition in theform of a capsule or tablet.
 3. The method of claim 1, wherein less than30% of the peptide is released upon exposure for 60 minutes to simulatedgastric fluid having a pH of 5 or less.
 4. The method of claim 1,wherein the delayed release coating is about 20% by weight of the beadwith the peptide coating.
 5. The method of claim 4, wherein the beadscomprise one or more additional coatings.
 6. The method of claim 1,wherein the beads have an average size of from 5 to 30 mesh.
 7. Themethod of claim 5, wherein the beads have one or more additionalcoatings selected from a base coat, a separating layer, and an overcoatlayer.
 8. The method of claim 1, wherein the composition furthercomprises a second plurality of beads comprising a coating containingthe peptide, and comprising a pH-dependent coating to effect release ofthe peptide in the jejunum of the patient.
 9. The method of claim 8,wherein the second plurality of beads releases the peptide about 30minutes after the beads releasing peptide in the duodenum.
 10. A methodfor treating a celiac patient, comprising: administering a peptidehaving the amino acid sequence of SEQ ID NO: 1 to the patient, whereinthe peptide is formulated as an oral dosage, delayed-release compositionthat contains: a first population of peptide-coated beads having adelayed release coating that is a 1:1 co-polymer of methacrylic acid andethyl acrylate and which is stable in gastric fluid and unstable in nearneutral to alkaline environments so as to release peptide in theduodenum, and wherein at least 70% of the peptide in said beads isreleased upon exposure for 90 minutes to simulated intestinal fluidhaving a pH of greater than 5; and a second population of peptide-coatedbeads having a delayed release coating that is a 1:1 co-polymer ofmethacrylic acid and ethyl acrylate and which is stable in gastric fluidand unstable in near neutral to alkaline environments so as to releasepeptide in the jejunum, and wherein at least 70% of the peptide in saidbeads is released about 30 minutes after the first population insimulated intestinal fluid having a pH of greater than 5; and whereinthe beads contain 0.1 to 0.8% by weight of said peptide.
 11. The methodof claim 1, wherein the amount of peptide administered is about 1.0 μgto about 1,000 μg.
 12. The method of claim 11, wherein the amount ofpeptide administered is not within the range of 1.0 μg to 100 μg. 13.The method of claim 10, wherein the amount of peptide administered isabout 1.0 μg to about 1,000 μg.
 14. The method of claim 13, wherein theamount of peptide administered is not within the range of 1.0 μg to 100μg.